This application is a continuation of U.S. patent application Ser. No. 09/643,206 filed on Aug. 21, 2000, now U S. Pat. No. 6,632,628, which claims priority to U.S. Provisional Application No. 60/150,048 filed on Aug. 20, 1999, the entire contents of each which are incorporated herein by reference. Additionally, all patents, published patent applications, and other references cited throughout this specification are hereby incorporated by reference in their entireties.
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns the discovery of a central mediator of cardiac hypertrophy.
2. Description of Related Art
Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrythmias, endocrine disorders and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy, heart failure, and sudden death. In the United States, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%.
Despite the diverse stimuli that lead to cardiac hypertrophy, there is a prototypical molecular response of cardiomyocytes to hypertrophic signals that involves an increase in cell size and protein synthesis, enhanced sarcomeric organization, upregulation of fetal cardiac genes, and induction of genes such as c-ƒos and c-myc (reviewed in Chien et al., 1993; Sadoshima and Izumo, 1997). The causes and effects of cardiac hypertrophy have been documented extensively, but the underlying molecular mechanisms that couple hypertrophic signals, initiated at the cell membrane to reprogram cardiomyocyte gene expression remain poorly understood. Elucidation of these mechanisms is a central issue in cardiovascular biology and is critical in the design of new strategies for prevention or treatment of cardiac hypertrophy and heart failure.
Numerous studies have implicated intracellular Ca2+ as a signal for cardiac hypertrophy. In response to myocyte stretch or increased loads on working heart preparations, intracellular Ca2+ concentrations increase (Marban et al., 1987; Bustamante et al., 1991; Hongo et al., 1995). This is consistent with a role of Ca2+ in coordinating physiologic responses with enhanced cardiac output. A variety of humoral factors, including angiotensin II (AngII), phenylephrine (PE) and endothelin-1 (ET-1), which induce the hypertrophic response in cardiomyocytes (Karliner et al., 1990; Sadoshima and Izumo, 1993a, 1993b; Leite et al., 1994), also share the ability to elevate intracellular Ca2+ concentrations.
Hypertrophic stimuli result in reprogramming of gene expression in the adult myocardium such that genes encoding fetal protein isoforms like β-myosin heavy chain (MHC) and α-skeletal actin are upregulated, whereas the corresponding adult isoforms, α-MHC and α-cardiac actin, are downregulated. The natriuretic peptides, atrial natriuretic factor (ANF) and β-type natriuretic peptide (BNP), which decrease blood pressure by vasodilation and natriuresis, also are rapidly upregulated in the heart in response to hypertrophic signals (reviewed in Komuro and Yazaki, 1993). The mechanisms involved in coordinately regulating these cardiac genes during hypertrophy are unknown, although binding sites for several transcription factors, including serum response factor (SRF), TEF-1, AP-1, and SpI are important for activation of fetal cardiac genes in response to hypertrophy (Sadoshima and Izumo, 1993a; 1993b; Kariya et al., 1994; Karns et al., 1995; Kovacic-Milivojevic et al., 1996). Most recently, the cardiac-restricted zinc finger transcription factor GATA4 also has been shown to be required for transcriptional activation of the genes for Ang II type 1α receptor and β-MHC during hypertrophy (Herzig et al., 1997; Hasegawa et al., 1997; reviewed in Molkentin and Olson, 1997).
The potential roles of the myocyte enhancer factor-2 (MEF2) family of transcription factors in cardiac development and hypertrophy are also considered. There are four members of the MEF2 family, referred to as MEF2A, -B, -C, and -D, in vertebrates (reviewed in Olson et al., 1995). These transcription factors share homology in an N-terminal MADS-box and an adjacent motif known as the MEF2 domain. Together, these regions mediate DNA binding, homo- and heterodimerization, and interaction with various cofactors, such as the myogenic bHLH proteins in skeletal muscle. MEF2 binding sites, CT(A/T)4TAG/A, are found in the control regions of the majority of skeletal, cardiac, and smooth muscle genes. The C-termini of the MEF2 factors function as transcription activation domains and are subject to complex patterns of alternative splicing.
During mouse embryogenesis, the MEF2 genes are expressed in precursors of cardiac, skeletal and smooth muscle lineages and their expression is maintained in differentiated muscle cells (Edmondson et al. 1994). The MEF2 factors also are expressed at lower levels in a variety of nonmuscle cell types. Targeted inactivation of MEF2C has been shown to result in embryonic death at about E9.5 due to heart failure (Lin et al., 1997). In the heart tubes of MEF2C mutant mice, several cardiac genes fail to be expressed, including α-MHC, ANF, and α-cardiac actin, whereas several other cardiac contractile protein genes are expressed normally, despite the fact that they contain essential MEF2 binding sites in their control regions. These results have demonstrated the essential role of MEF2C for cardiac development and suggest that other members of the MEF2 family may have overlapping functions that can support the expression of a subset of muscle genes in the absence of MEF2C. In Drosophila, there is only a single MEF2 gene, called D-MEF2. In embryos lacking D-MEF2, no muscle structural genes are activated in any myogenic lineage, demonstrating that MEF2 is an essential component of the differentiation programs of all muscle cell types (Lilly et al., 1995; Bour et al., 1995).
Although MEF2 factors are required for activation of muscle structural genes, they are not sufficient to activate these genes alone. Instead, biochemical and genetic studies have shown that MEF2 factors act combinatorially with other transcription factors to activate specific programs of gene expression. In skeletal muscle, MEF2 establishes a combinatorial code through interaction with members of the MyoD family to activate muscle gene transcription (Molkentin et al., 1995; Molkentin and Olson, 1996). The specific partners for MEF2 in cardiac and smooth muscle cells or in nonmuscle cells in which MEF2 proteins have been shown to regulate a variety of genes, remain to be defined.
As discussed below, there are four lines of evidence that suggest an important role for MEF2 in the control of cardiac hypertrophy. 1) MEF2 regulates many of the fetal cardiac genes that are up-regulated during hypertrophy. 2) MEF2 transcriptional activity is induced by the same signal transduction pathways that control hypertrophy. 3) MEF2C is upregulated in the hearts of human patients with congestive heart failure. 4) MEF2 synergizes with the thyroid hormone receptor to regulate transcription of the α-MHC gene (Lee et al., 1997) and thyroid hormone is a potent inducer of hypertrophy.
Transcriptional activation of the orphan steroid receptor Nur77 gene (NGFI-B) in T cells in response to T cell receptor activation is mediated by a CsA-sensitive, calcium-dependent signaling pathway (Woronicz et al., 1995). This signaling pathway is directed at two MEF2 binding sites in the NGFI-B promoter. There is no change in DNA binding activity of MEF2 in the presence or absence of calcium signals in that system, whereas transcriptional activity of MEF2 is dramatically increased by calcium signaling. This implies that calcium signals must enhance MEF2 activity by inducing a cofactor or a posttranslational modification of MEF2 that stimulates transcriptional activity.
In addition, transcription of the calcium-dependent lytic cycle switch gene BZLF1, which is required for induction of the lyric cycle of Epstein-Barr virus (EBV), is inhibited by CsA and FK506, indicating that a calcineurin-dependent pathway mediates activation of this gene (Liu et al, 1997). CsA-sensitivity of BZLF1 transcription maps to three MEF2 sites in the BZLF1 promoter. CsA-sensitive inducibility was shown to be reconstituted using an artificial promoter containing multiple copies of the MEF2 site in conjunction with a CREB/AP-1 site. NFAT did not bind the BZLF1 promoter, but CsA-sensitive induction of this promoter was shown to be calcineurin- and NFAT-dependent. CaMKIV was also shown to be a potent inducer of MEF2 activity (Liu et al., 1997). The mechanism whereby MEF2 confers responsiveness to the calcineurin/NFAT signaling system remains to be elucidated, however.
The MAP kinase signaling pathway also has been shown to lead to enhanced transcriptional activity of MEF2 factors in a variety of cell types (Han et al., 1997; Coso et al., 1997; Kato et al., 1997; Clarke et al., 1998). This enhancement has been shown for MEF2C to be mediated by phosphorylation of three amino acids, Thr293, Thr300, and Ser387, in the C-terminal activation domain by the MAP kinase family member p38. Whether these same residues are phosphorylated by hypertrophic signaling in the heart remains to be determined.
It is clear that the cardiac hypertrophic response is somehow initiated through a Ca2+ dependent pathway. However, the precise identification of the gene(s) which mediate(s) the hypertrophic response remains elusive. The present invention is directed toward the elucidation of the exact point in the hypertrophic pathway which may be manipulated to achieve beneficial effects on cardiac hypertrophy. In order to develop pharmacologic strategies for treatment of cardiac hypertrophy in humans, it will be important to establish experimental models which accurately reflect the pathological profile of the disease and to identify compositions which regulate or inhibit hypertrophic growth.